Campylobacter isolates were identified and further characterized. The species was determined via MALDI-TOF MS (Autoflex Speed LRF, Bruker, Billerica, MA, USA) [27 (link)], as described above (Section 2.4 ). Hereafter, clonal complexes (CC) and sequence types (ST) of the strains were determined via multilocus sequence typing (MLST). Via the DNeasy Blood and Tissue kit (Qiagen, Venlo, Hilden, Germany), DNA was extracted from the isolates, and the DNA yield and quality were examined spectrophotometrically (NanoDrop, Thermo Scientific, Waltham, MA, USA). Finally, the samples were diluted to 25 ng/µL. Primers, PCR conditions, sequencing and MLST scheme were used as described by Jolley et al. [28 (link)]. The isolated strains were analyzed via MLST to determine whether the strains colonizing broiler flocks were homologous or heterologous strains compared to those colonizing the respective breeder flocks.
Autoflex speed lrf
Manufactured by Bruker
Sourced in United States, Germany
The Autoflex Speed LRF is a laser-based matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer produced by Bruker. It is designed for high-throughput analysis of a wide range of sample types, including proteins, peptides, and small molecules.
Automatically generated - may contain errors
Lab products found in correlation
Pierce trypsin protease, by Thermo Fisher Scientific (2 mentions)
Flexanalysis 3, by Bruker (2 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Jem 1011, by JEOL (1 mentions)
Protein standard 1, by Bruker (1 mentions)
Maldi tof ms, by Bruker (1 mentions)
9 protocols using autoflex speed lrf
1
Campylobacter Strain Characterization in Broiler Flocks
2
Zooarchaeological Collagen Fingerprinting Protocol
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Analysis was carried out at the ZooMS facility of the Department of Archaeology at the Max Planck Institute for the Science of Human History, Jena, Germany. We followed established protocols.27 ,28
In brief, from each bone approximately 10-20 mg was removed using a circular diamond drill-bit. Samples were rinsed in ammonium bicarbonate overnight and incubated for 1h at 65°C. The supernatant was treated with 0.4 μg trypsin (Thermo Scientific Pierce™ Trypsin Protease) and allowed to digest at 37 °C for 18 h. The incubated samples were concentrated and desalted using C18 ZipTips (Thermo Scientific Pierce™ C18 Tips) and eluted in a final solution of 50μl of 50% acetonitrile and 0.1% TFA. 0.5 μl of the resulting solution was mixed with 0.5 μl of α - cyano-4-hydroxycinnamic acid solution (10 mg/mL in 50% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA) and allowed to crystallize. The samples were analysed using a MALDI TOF/TOF (Bruker Autoflex Speed LRF) mass spectrometer. The resulting spectra were screened for diagnostic markers using flexAnalysis 3.4 (Bruker Daltonics) and mMass software.41 (link)
The spectra were compared against a reference library of known peptide markers.12 (link),15 (link),30
In brief, from each bone approximately 10-20 mg was removed using a circular diamond drill-bit. Samples were rinsed in ammonium bicarbonate overnight and incubated for 1h at 65°C. The supernatant was treated with 0.4 μg trypsin (Thermo Scientific Pierce™ Trypsin Protease) and allowed to digest at 37 °C for 18 h. The incubated samples were concentrated and desalted using C18 ZipTips (Thermo Scientific Pierce™ C18 Tips) and eluted in a final solution of 50μl of 50% acetonitrile and 0.1% TFA. 0.5 μl of the resulting solution was mixed with 0.5 μl of α - cyano-4-hydroxycinnamic acid solution (10 mg/mL in 50% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA) and allowed to crystallize. The samples were analysed using a MALDI TOF/TOF (Bruker Autoflex Speed LRF) mass spectrometer. The resulting spectra were screened for diagnostic markers using flexAnalysis 3.4 (Bruker Daltonics) and mMass software.41 (link)
The spectra were compared against a reference library of known peptide markers.12 (link),15 (link),30
3
Quantifying Propranolol in Mouse Kidney
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Example 3
A mouse kidney treated with 7.5 mg/kg of propranolol, and sacrificed 20 minutes post-administration, is taken.
Three 20 μm thick tissue slices are produced from the kidney and are each deposited on a support (slide ITO).
MALDI DHB matrix (40 mg/ml methanol/TFA 0.1% 1/1) is sprayed using a robotized system (SunCollect) onto the slices.
Each slice is analysed by mass spectrometry imaging, using an Autoflex speed LRF (Bruker, Daltonics). The average intensity of the peaks of the mass spectrum of the propranolol (m/z 260.2) is computed, by once again using a number of ROIs on each slice, all dimensions being the same.
The average intensity value obtained is 19633.3.
The propranolol concentration in the kidney is quantified using the calibration curve equation (
4
Synthesis and Characterization of Methyltetrazine-Conjugated Peptides
5
MALDI-TOF-MS Analysis of Abx-BM(PEG)2 and IL4R-Abx
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The molecular weight of Abx-BM(PEG)2 and IL4R-Abx was determined using matrix-assisted laser desorption-ionization/time of flight mass spectrometry (MALDI-TOF-MS). MALDI-TOF-MS analyses were conducted using an Autoflex speed LRF, Bruker (smart beam-II laser (355 nm)) operating in linear mode/positive ion mode with a mass-to-charge (m/z) ratio range of 30-210 kDa. The laser fluence was set at 90%, with a laser frequency of 2000 Hz, and more than 10000 laser shots accumulated. The sample (4 mg/mL) was dissolved in an aqueous solution containing 50% acetonitrile (ACN) with 0.1% TFA (trifluoroacetic acid). Sinapic acid was served as the matrix. The hydrodynamic size of nanoparticle was measured by dynamic light scattering (DLS) with Zetasizer Nano S90 systems (Malvern, UK). All groups were dissolved at a concentration of 10 mg/mL in PBS buffer. To examine structural feature, transmission electron microscopy (TEM) images were obtained using JEM-1011 instrument (JEOL Ltd, Japan), and analyzed with Gatan digital microscope software. All groups are dissolved at a concentration of 0.01 mg/mL in deionized water.
6
Zooarchaeological Collagen Fingerprinting Protocol
7
MALDI-TOF MS Protein Analysis Protocol
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For MALDI-TOF MS analysis, proteins were extracted by using an extended direct transfer method that included a formic acid overlay (36 (link), 37 ). Measurements were performed in linear and reflectron modes by using an Autoflex Speed LRF mass spectrometer (Bruker Daltonics) equipped with a smartbeam-II laser (355 nm). Detailed instrument settings are available in SI Appendix .
8
MALDI-TOF MS Analysis of ATIs
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ATIs were analyzed for their molecular weight with a MALDI-TOF MS instrument (Autoflex Speed LRF, Bruker Daltonik GmbH, Bremen, Germany) [43 (link)]. 2 µL of samples were mixed with 2 µL of 2% TFA and 2 µL of 2,5-Dihydroxy actetophenone (Alfa Aesar, Heysham, UK) and aliquots of 2 µL were applied to the target plate (MTP target frame III, Daltonik). Protein Standard I (Bruker Daltonics) containing a mixture of four proteins was used for internal calibration and the measurements were performed in linear mode (LM).
9
MALDI-TOF-MS Analysis of Archaeological Samples
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Modern reference samples were spotted in triplicate onto an MTP AnchorChip 384-target plate, together with matrix solution (10 mg of α-cyano-4-hydroxycinnamic acid in 7 ml of 85% acetonitrile (ACN)/0.1% trifluoracetic acid (TFA)). Archaeological samples were mixed with matrix solution (α-cyano-4-hydroxycinnamic acid of 10 mg ml−1 in 50% ACN/0.1% TFA) and spotted onto an MTP Groundsteel 384-target plate. All samples were analysed using an Autoflex Speed LRF matrix-assisted laser desorption/ionization–tandem time of flight mass spectrometer (MALDI-TOF-MS, Bruker Daltonics) with a smartbeam-II laser. A SNAP averaging algorithm was used to obtain monoisotopic masses (C: 4.9384, N: 1.3577, O: 1.4773, S: 0.0417, H: 7.7583).
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